JPH0742319B2 - Polypeptide having motilin-like activity and use thereof - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel polypeptide having motilin-like activity and its use.

The polypeptides according to the invention can be used as pharmaceutical ingredients, in particular for the treatment of gastrointestinal disorders.

(Prior Art) Motilin was a substance whose structure was determined and isolated for the first time from the upper small intestinal mucosa of pigs by Brown et al., And is a kind of peptide hormone ["Gastroenterology" Vol. 62, pp. 401-404 ( 1972) and "Can.J.Biochem." Vol. 52, pp. 7-10 (1974)]. This porcine motilin is composed of 22 amino acids,
The molecular weight is about 2700.

On the other hand, regarding motilin of human origin, its cDNA clone was isolated and its structure was determined by the present inventors, and its amino acid sequence was clarified to be the same as that of porcine origin (JP-A-63-63). 276489)].

As the physiological actions of motilin, gastrointestinal motility enhancement action and digestive tract smooth muscle contraction action are well known. Among these actions, as for the action of promoting gastrointestinal motility, for example, an action of shortening the excretion time of the contents in the stomach has been reported [“Gastroenterology”, Vol. 80, pp. 456-460 (1981).
)], And it is known that the gastrointestinal smooth muscle contraction effects strong contraction on the gastric antrum and duodenum of rabbits and humans without depending on the neural pathway. Since motilin enhances gastrointestinal motility and no particular side effects have been reported, it has been considered that motilin is effective for treatment of gastrointestinal disorders such as post-surgery and diagnosis of gastrointestinal disorders. For treatment of ileus (intestinal palsy) etc. later,
Conventionally, prostaglandin F ₂ α and the like have been generally used, but there is a problem in that side effects are strong].

The 13th position in the amino acid sequence for motilin is methionine, but it has been reported that a polypeptide having an amino acid structure in which it is converted to norleucine or leucine also shows the same physiological activity as motilin [“Scad. J. Gas
troenterology "Vol. 11, pp. 119-203 (1976), etc.], and it is therefore considered that methionine at the 13th position has almost no effect on the activity.

(Problems to be Solved by the Invention and Objects of the Invention) According to a conventional technique, motilin is generally derived from pigs, obtained by extraction, and mass production was extremely difficult. On the other hand, even when the chemical synthesis method is used, it is difficult to obtain motilin in large quantities and at low cost, since motilin is a polypeptide consisting of 22 constituent amino acids.
That is, although motilin is expected to be useful in the treatment and diagnosis of gastrointestinal disorders, its productivity has become a bottleneck and has not been widely used for clinical applications.

Therefore, so-called “biotechnology” has been applied to research to inexpensively produce a polypeptide having a motilin-like physiological activity (Japanese Patent Laid-Open No. 63-71195 and Japanese Patent Laid-Open No. 63-71195). 2-57192, etc.).

In the above method utilizing biotechnology,
It has a step of cleaving with bromocyan, and an operation of removing an extra peptide generated in this case and containing a homoserine residue on the C-terminal side by an enzymatic treatment was carried out.

Furthermore, studies have also been conducted on amino acid structural modifications showing an activity equal to or higher than motilin (Japanese Patent Publication No. 61-2).
6559 bulletin etc.).

The main object of the present invention, which should be developed in consideration of the above-mentioned known techniques and related techniques, is that they have physiological activity equal to or higher than motilin and are easy to prepare, that is, they can be obtained by cleaving with bromocyan. Therefore, it is to provide a motilin-like polypeptide (motilin analog, or “motilin analog”) that can be prepared without the need for enzymatic treatment.

An incidental but important object of the present invention is to provide a drug containing the above-mentioned polypeptide as an active ingredient, that is, a therapeutic agent for digestive tract disorders, which can be efficiently and inexpensively provided.

(Means and Actions for Solving the Problem and Achieving the Purpose) As a result of intensive studies made by the present inventors, regarding the polypeptide obtained by the fermentation method and the 13th position is an amino acid residue other than methionine (Met), After cleaving with bromocyan, even if the C-terminal part is not further treated with an enzyme,
It was found that the polypeptide itself obtained by cleavage with bromocyanine has an activity equal to or higher than motilin, and that a motilin analog obtained by converting an amino acid in a part thereof also has activity equal to or higher than motilin. The present invention has been completed, and the present invention has basically been completed.

Therefore, from one viewpoint, the present invention is based on the general formula (I ′) Phe-Z ₁ -A ₁ -Z ₂ -B ₂ -Thr-B ₃ -A ₂ -Glu-Z ₃ -C-Arg-X. -Gln-Glu-L
ys-Glu-Arg-D-Lys-Gly-E-Hse [wherein Z ₁ means Val or Leu, Z ₂ means lle or Leu, Z ₃ means Leu or lle, A ₁ Means Pro or Gly, A ₂ means Gly or Ser, B ₂ and B ₃ mean Phe or Tyr, C means Gln, Glu or Asp, X means Leu, D Means Asn, Glu or Asp, E means Gln, Lys or A
rg, and Hse means homoserine or homoserine lactone] and a salt thereof, which solves the above-mentioned problems and achieves the above-mentioned main purpose. .

In the above general formula (I ′), the amino acid at position 1 is phenylalanine (Phe), but the amino acid residue at this position is tyrosine (Tyr), which is an amino acid having a side chain having an aromatic ring like Phe. ) Has proved to be all right.

Thus, the polypeptide according to the invention is essentially of the general formula (I) B ₁ -Z ₁ -A ₁ -Z ₂ -B ₂ -Thr-B ₃ -A ₂ -Glu-Z ₃ -C-Arg. -X-Gln-Glu-Ly
s-Glu-Arg-D-Lys-Gly-E-Hse [wherein Z ₁ means Val or Leu, Z ₂ means lle or Leu, Z ₃ means Leu or lle, A ₁ Means Pro or Gly, A ₂ means Gly or Ser, B ₁ , B ₂ and B ₃ are Phe or T
means yr, C means Gln, Glu or Asp, X means Leu, D means Asn, Glu or Asp, E means Gln, Lys
Or Arg, and Hse means homoserine or homoserine lactone].

The above-mentioned additional objects are achieved by a therapeutic agent for gastrointestinal tract disorders containing the polypeptide represented by the above general formula (I) or a salt thereof as an active ingredient.

In the polypeptide represented by the above general formula (I), Va, which is an amino acid residue represented by Z ₁ , Z ₂ and Z _3.
l, Leu and lle have in common that they have a hydrophobic side chain, and Pr which is the amino acid residue that A ₁ and A ₂ mean.
o, Gly and Ser are common in that they can have a bent structure in the peptide chain, and B ₁ , B ₂ and B ₃ mean amino acid residues Phe and Tyr have an aromatic ring. They have in common that they have side chains.

(Examples, etc.) Next, the present invention will be described in more detail with reference to production examples, pharmacological activity tests and formulation examples.

Production Example Peptide synthesis was carried out using a peptide synthesizer 430A manufactured by Applied Biosystems.

For example, a polypeptide having the amino acid sequence shown below In the case of synthesizing, the polypeptide having the following amino acid sequence To synthesize.

Purification of the synthesized polypeptide sample was performed using Waters Microbonder C-18 column (19 mm × 15 c).
m) was carried out by HPLC under the following conditions.

Eluent: linear gradient of 30% to 60% acetonitrile in 0.1% trifluoroacetic acid for 30 minutes Flow rate: 7.0 ml / min Purified sample 10 mg is weighed and dissolved in 70% formic acid solution 30 ml, 50 mg bromcian is added, The reaction was carried out at 37 ° C for 16-24 hours. Next, 200 ml of distilled water was added and freeze-dried to remove formic acid and bromocyan. About this again Waters Micro Bonder Sphere
Purification by HPLC using a C-18 column (19 mm x 15 cm) and under the same conditions as above.

The main peak portion by this HPLC was recovered, freeze-dried, and a part thereof was examined by a peptide sequencer manufactured by Applied Biosystems. As a result, the sample was correctly cleaved at the position of methionine (Met) and the C-terminal was homoserine (Hse). ) Was produced.

After synthesizing a polypeptide in which a part of the constituent amino acids has been variously converted with a peptide synthesizer in the same manner as above,
It could be obtained by purification by HPLC.

Physiological activity test example 1 (activity measurement of [L-Leu ¹³ ] -motilin-Hes and salts thereof) The polypeptide obtained in Production Example, wherein the 23rd position is homoserine (Hse) ([L- Leu ¹³ ] -motilin-Hse) was used as a test substance, while it was chemically synthesized [L
-Leu ¹³ ] -Motilin was used as a control substance, and the intestinal contractile activity at the time of administration of 2.0 μg / kg / hr was measured by a balloon method using a living organism of dogs. The results are shown in FIGS. 1A and 1B. .

As is clear from FIGS. 1A and 1B, the polypeptide according to the present invention ([L-Leu ¹³ ] -motilin-Hse) was compared with known [L-Leu ¹³ ] -motilin in intestinal contractile activity. It was confirmed that the intestinal contractile activity was extremely spontaneous and that it was significantly spontaneous.

In addition, it was confirmed that the pharmacological activity of homoserine at the 23-position forming a lactone ring was equivalent to that of homoserine type.

Furthermore, [L-Leu ¹³ ] -motilin-Hse and its sodium salt were used as test substances 1 and 2, and the intestinal contractile activity was measured by the balloon method using a dog living body in the same manner as described above,
The contractile activity in the duodenum at the time of administration of 2.0 μg / kg / hr was determined, and the results of comparison with the contractile activity of L-Leu ¹³ ] -motilin as a control substance taken as 100% are shown in Table 1 below. Both test substances are 10-20 more than the control substances
It was found that the activity was as high as about%.

Furthermore, the intestinal contractile activity of prostaglandin F ₂ α, which is a known substance having intestinal contractile activity and which is currently clinically used for the treatment of postoperative Reus, is measured by the balloon method in the same manner as above. However, the activity of the drug and the polypeptide of the prior art [L-Leu ¹³ ]-
The results of comparison between the activity of motilin and the activity of the polypeptide of the present invention L-Leu ¹³ ] -motilin-Hse and its sodium salt are shown in FIG. 2, and the polypeptide of the present invention is It has been found that the drug is active at a much lower concentration when compared with the currently widely used drug, prostaglandin F ₂ α, and therefore the dose can be kept extremely low.

Taking these facts into consideration and as is apparent from FIGS. 1A and 1B, the polypeptide according to the present invention, in particular [L-Le
Since u ¹³ ] -motilin-Hse shows a more spontaneous intestinal contractile action when compared with [L-Leu ¹³ ] -motilin,
It can be said that the present invention provides a polypeptide that can be used as a drug far superior to existing drugs in terms of use safety and action.

Physiological Activity Test Example 2 (Activity Confirmation of Polypeptides Partially Converting Motilin Constituent Amino Acids) For polypeptides in which part of motilin constituent amino acids was converted, Magnus method using rabbit duodenum [“J.Ph
rm.Pharmac. ", Vol.28.pages 650-651 (1976)], the intestinal contractile activity was measured, and ED50 was calculated for each, and the results of comparing motilin activity to 100% are shown in Table 2 below. It was on the street.

In Table 2, the C-terminal is represented by Hse, but the activity was almost the same even when this portion was in the form of homoyaline, homoserine lactone or sodium salt.

Regarding human motilin, it is said that its active center is located in the amino acid sequence near the N-terminal, but there is the following law between the amino acid near the N-terminal and the expression level of activity, and therefore the amino acid in this part is It was found from the results shown in Table 2 that the conversion of A.

(A) Motilin Pro at the 3rd position and Gly at the 8th position
, It is relatively easy to take a bent structure in the peptide chain
Motilin analogs converted to Gly, Ser or Asn show activity equal to or higher than that of motilin, but motilin analogs converted to lle, which is difficult to have a bent structure, show a marked decrease in activity.

In the above case, the motilin analog in which the 3-position is converted to Gly has extremely high activity, which is almost twice that of the original motilin.

(B) Val at the 2nd position of motilin, lle at the 4th position and Leu at the 10th position are amino acids having hydrophobic side chains, and motilin analogs having a structure in which these amino acids are mutually replaced are also equivalent to or equivalent to motilin. The above activities are shown.

(C) Phe at position 1 and 5 of motilin and Tyr at position 7 are amino acids having side chains having an aromatic ring, but motilin analogs having a structure in which these amino acids are mutually substituted have activity equivalent to that of motilin. However, the activity of motilin analogs converted to other amino acids, such as lle, is significantly reduced.

(D) Regarding the C-terminal side of motilin, it is composed of an amino acid having a charge or polarity, although it is not the active center,
Particularly, it is said to be important for maintaining the activity in the living body.
Therefore, as a related motilin analog to be used for the test, one having an amino acid structure in which attention was paid so as to increase the positive or negative charge at the C-terminal side was prepared, and the activity was investigated. Not seen and therefore, if the overall charge balance is retained, some amino acid conversions are presumed to have little effect on activity.

Formulation Example Polypeptide according to the present invention (Production Example [L-Leu ¹³ ]
-Motilin-Hse) was dissolved in purified water, aseptically dispensed into a vial so as to contain 1 mg of polypeptide, freeze-dried to remove water, and sealed to obtain a dry powder.

This agent is dissolved in physiological saline for injection or the like at the time of use.

(Effects of the Invention) The polypeptide according to the present invention is a known motilin or [L-Le
u ¹³ ] -has physiological activity equivalent to or higher than motilin.

The motilin analog according to the present invention is structurally at the C-terminal side.
It is characterized by having Hse (homoserine or homoserine lactone residue), which eliminates the need for the conventional enzyme treatment step in the preparation of a polypeptide, and as a result, naturally reduces the production cost, Manufacturing time is reduced. The motilin analog according to the present invention is genetically engineered, for example, by utilizing the method previously developed by the present inventors (see Japanese Patent Application Laid-Open No. 2-57192), that is, the motilin analogs are bound to each other in tandem. Amino acid is, for example, Met-Gly-lle-Phe-Met, and DNA encoding such an amino acid sequence is incorporated into a vector such as prosmid as genetic information and expressed in a host such as Escherichia coli, and then treated with bromocyan. Can be easily adjusted.

[Brief description of drawings]

FIG. 1A is a waveform diagram depicting changes in intestinal motility upon administration of [L-Leu ¹³ ] -motilin-Hse, which is a polypeptide of the present invention, by a balloon method using a living canine,
FIG. 1B is similar to FIG. 1A, but with reference material [L-Leu
^13] - is a waveform diagram when administered motilin, FIG. 2 [L-Leu ^13] - a polypeptide according to the motilin as well as the invention [L-Leu ^13] - motilin -Hse and its sodium salt and various ₂ is a graph showing the intestinal contractile activity shown at the concentration of 10% by comparison with the intestinal contractile activity shown by prostaglandin F ₂ α, which is a known drug.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Kenichi Tanaka 35 Higashi-Tobori-cho, Higashi-ku, Nagoya-shi, Aichi Stock Company Sanwa Chemical Research Institute (72) Inventor Katsuya Fujimura 35 Higashi-Tobori-cho, Higashi-ku, Nagoya, Aichi Stock Company Sanwa Chemical Research Institute (72) Inventor Yuji Hayashi 35, Higashi Sotobori-cho, Higashi-ku, Nagoya, Aichi Stock Company Sanwa Chemical Research Institute (72) Inventor Yohei Kobayashi 35 Higashi-Tobori-cho, Higashi-ku, Aichi Prefecture Sanwa Co., Ltd. Inside the Chemical Research Laboratory (72) Inventor Kiichi Sawai, 35 Higashi Sotobori-cho, Higashi-ku, Nagoya, Aichi (56) Inside the Sanwa Chemical Research Institute, Inc. (56) Reference JP 63-71195 (JP, A) JP 54-9267 ( JP, A) JP 52-73863 (JP, A) JP 52-46068 (JP, A) JP 49-135964 (JP, A)

Claims (2)

[Claims] 1. General formula (I) B ₁ -Z ₁ -A ₁ -Z ₂ -B ₂ -Thr-B ₃ -A ₂ -Glu-Z ₃ -C-Arg-X-Gln-Glu-Ly
s-Glu-Arg-D-Lys-Gly-E-Hse [wherein Z ₁ means Val or Leu, Z ₂ means lle or Leu, Z ₃ means Leu or lle, A ₁ Means Pro or Gly, A ₂ means Gly or Ser, B ₁ , B ₂ and B ₃ are Phe or T
means yr, C means Gln, Glu or Asp, X means Leu, D means Asn, Glu or Asp, E means Gln, Lys
Or Arg, and Hse means homoserine or homoserine lactone] and a salt thereof. 2. General formula (I) B ₁ -Z ₁ -A ₁ -Z ₂ -B ₂ -Thr-B ₃ -A ₂ -Glu-Z ₃ -C-Arg-X-Gln-Glu-Ly
s-Glu-Arg-D-Lys-Gly-E-Hse [wherein Z ₁ means Val or Leu, Z ₂ means lle or Leu, Z ₃ means Leu or lle, A ₁ Means Pro or Gly, A ₂ means Gly or Ser, B ₁ , B ₂ and B ₃ are Phe or T
means yr, C means Gln, Glu or Asp, X means Leu, D means Asn, Glu or Asp, E means Gln, Lys
Or Arg, and Hse means homoserine or homoserine lactone].